Generation and use of new types of dendritic cells

ABSTRACT

The present invention relates to a method for the differentiation and/or maturation of immature myeloid dendritic cells (DC) into HLA-DR, CD86, CD83 and IL12 (p40) dendritic cells comprising incubating said DC with dg T cells; and to a method for the differentiation and/or maturation of immature myeloid dendritic cells (DC) into HLA-DR, CD86, CD83 and IL12 (p70) dendritic cells comprising incubating said DC with activated dg T cells. Specific compositions and uses thereof are described.

TECHNICAL FIELD

The present invention relates to the improvement of the therapy for thetreatment or prevention of cancer, infections and autoimmune diseases inparticular in the development of new dendritic cells carrying a superiorcharacter in inducing T cell responses.

BACKGROUND ART

For years people try to understand the regulatory system of theimmunological response. It is clear that the immune reaction resultsfrom a complex interaction between cellular and humoral responses.

Dendritic cells (DC) represent the most important antigen presentingcells for the induction of primary T cell responses (1). In order toefficiently exert their function in lymphoid organs, DC have to undergoa maturation process which is initiated in peripheral tissues.Maturation of DC results in the expression of high levels MHC andcostimulatory molecules on their membrane and is often associated withthe secretion of interleukin (IL)-12 (2-3), a critical factor for thedevelopment of Th1-type responses.

As this cell type is endowed with the unique capacity to cluster naïve Tcells, it has been proposed as a natural adjuvant, aiming at thetriggering of T-cell responses against poor immunogens, such astumor-associated Ag (TAA). The realization of clinical trials has longbeen impaired by the low frequency of circulating DC available in theblood. The development of methods of generating large numbers of DC fromhemapoietic precursors has recently allowed the initiation of pioneerclinical trials. Human myeloid DC can be easily generated in vitro byculturing monocytes in presence of GM-CSF and IL-4 whereas the so-calledlymphoid DC have been obtained by isolation of precursors from blood orlymph nodes. Recently, the inventors described a new type of stabledendritic cell (DC) which is more mature and is more potent inactivating the immune system compared to other known stable DCs(EP00870273.0). Indeed, monocytes cultured in IL3 and IFN-βdifferentiate into IL3-R+ CD11c+ myeloid dendritic cells with potent Tcell stimulatory activities.

The GM-CSF/IL4 DC have been used in clinical trials in cell therapy.However, using these conditions, only 67% of immunized patients showedan increase in response to the treatment. Therefore it is important toimprove the method, conditions or substances to improve the efficacityof the treatment.

OBJECTS OF THE INVENTION

The present invention is directed towards providing a method for theproduction of superiour dendritic cells which can be used in celltherapy to eliminate or prevent more efficiently the deleterious effectsof invasive cells in patients.

In contrast to the well studied influence of cytokines on the DCmaturation, less attention has been paid so far to the possiblecontribution of cells taking part in innate immune defenses to thiseffect; The inventors found surprisingly that dendritic cells, presentin an early developing stage (referred to as immature myeloid DCs inthis patent application) could be further differentiated and matured byincubating these with a specific type of T cell: the γδ T cells.

γδ T cells are rapidly activated by bacterial products and subsequentlyrelease cytokines such as TNF-α and interferon (IFN)-γ (6-9). Indeed,unlike classical αβT cells, γδ T cells have the ability to interact withnon-processed antigens (10). For human γδT cells expressing Vγ9 andVδ2-encoded receptors, major ligands are represented by phosphoantigenswhich stimulate their proliferation and their secretion of cytokines(11-15). Bromohydrin pyrophosphate (BrHpp) is a synthetic phosphoantigenwhich was recently shown to efficiently induce activation of humanVγ9/Vδ2 T cells (16).

The inventors found that especially these γδ T cells have a surprisingeffect on the activation of monocyte-derived DC. In addition, theinventors showed that the induction of said cells using BrHpp furtherincreased this stimulating effect. In this patent application, theinventors tried to unravel which molecules are mediating the γδT cell-DCinteractions.

The present invention identifies a new strategy to improve the abilityof DC to elicit T cell responses.

These aims have been met by following embodiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for the differentiation and/ormaturation of immature myeloid dendritic cells (DC) into HLA-DR, CD86,C083 and IL12 (p40) dendritic cells comprising incubating said DC withδγ T cells. With ‘immature myeloid dendritic cells’ it is meant cellswhich are cells of the myeloid lineage involved in antigen presentationincluding monocytes, other dendritic cell precursors and dendritic cellsthemselves. The effect of δγ T cells on DC was only studied for myeloidcells. Nevertheless, the inventors do not exclude the possiblility thatthe method for the differentiation and/or maturation of immature myeloidDC, as suggested in present patent application, can be applied for thedifferentiation and/or maturation of immature lymphoid DC. This has tobe interpreted throughout the whole patent application.

There are two main sources of DC precursors: CD34+ stem cells andperipheral blood (PB) monocytes. The main constraints of generating DCfrom stem cells is that the culture time is long and obtaining CD34+cells requires mobilization of the patient. Therefore a preferredembodiment of the present invention is to use monocytes as a DCprecursor. Respective DC precursor can be induced (see below) creatingimmature myeloid dendritic cells. Nevertheless, these immature myeloiddendritic cells are not fully mature and probably induce theimmunological response only partially when an antigen is present. Tocharacterize additional factors, the inventors investigated the effectof specific immune cells on the differentiation and/or maturation ofsaid cells.

The inventors found surprisingly that coculture of DC with γδ T cellsresulted in the upregulation of HLA-DR, CD86, CD83 surface markers,indicating that DC undergo some degree of maturation under the influenceof γδT cells.

In addition, freshly isolated γδT cells induced the production of IL-12(p40) but did not elicit IL-12 (p70) production. Therefore, theinventors defined the differentiated cells of present invention, usingδγ T cells, as HLA-DR, CD86, CD83 and IL12 (p40) dendritic cells.

Until now, δγ T cells are isolated from fresh blood. Nevertheless, forthe method as described by the invention, also in vitro induced γδ Tcells, or cells which can be differentiated into γδ T cells, can beused.

The inventors also points towards the fact that the method according topresent invention for the differentiation and/or maturation of immaturemyeloid dendritic cells (DC) into HLA-DR, CD86, CD83 and IL12 (p40)dendritic cells can be performed in vitro or in vivo. For example, γδ Tcells can be injected in patients resulting in the in vivo production ofHLA-DR, CD86, CD83 and IL12 (p40) dendritic cells. The inventors suggestthat these HLA-DR, CD86, CD83 and IL12 (p40) dendritic cells have ahigher capacity to induce efficient Th 1-type and CTL responses comparedto already known dendritic cells.

Preferentially, said immature myeloid DC are derived from monocytesthrough cytokine treatment chosen from IL-4/GM-CSF or IFN-β/IL-3 orfunctional analogues thereof. It has been previously shown thatmonocytes can be differentiated in myeloid dendritic cells using acombination of specific cytokines: IL-4 and GM-CSF, or, IFN-β and IL-3creating IL-4/GM-CSF DC, L-3/IFN-β DC, respectively. IL-3 and IFN-β DCexpress markers of the myeloid lineage (CD11c, CD14, and CD33) andinduce high levels of HLA class I and class II molecules, CD40, CD54, CD80 and CD86, and IL-3Rα (CD123). Contrary to IL-4/GM-CSF DC, IL-β/IFN-βDCs show much higher levels of IL-3Rα. Conversely, CD1a is expressed onIL-4/GM-CSF DC but not on IL-3/IFN-β DC.

According to the invention, said cytokines are added simultaneously,sequentially or separately with the δγ T cells to the monocytes. In theexamples, evidence shown that said immature myeloid DCs are furthermaturated by the γδ T cells interaction. Nevertheless, the inventors donot exclude the possibility that a similar result may be obtained byfirst contacting the monocytes with the γδ T cells prior to the cytokinetreatment. In addition, it is obvious for a person skilled in the artthat both steps may be performed simultaneously.

It is clear from present invention that specific cytokines, secreted bythe γδ T cells, make the DC differentiate. The inventors showed in theirexamples that TNFα and IFN-γ are responsible for observed effects,Nevertheless, the inventors have evidence that there are more factorsincluding interactions between membrane-bound molecules provided bythese γδ T cells which makes DC to differentiate further.

In the examples of present invention, a cell ratio of 1:1 for immaturemyeloid DC: γδ T-cells is used. It is obvious for a person skilled inthe art that, variation in this cell ratio is possible. One needs totake in account that, if only a small number of cells are provided, thematuration of these DC can not be performed anymore. This may beexplained by the fact that a minimal concentration of these secretedand/or cell factors are necessary to induce further differentiationand/or maturation of DC. Therefore the inventors suggest in the presentinvention to use a cell ratio of the δγ T cells over themonocytes/dendritic cells between 0.1 and 10. For example, the cellratio of the δ T cells over the monocytes/dendritic cells is 1:1.

The present patent application also describes a method for thedifferentiation and/or maturation of immature myeloid dendritic cells(DC) into HLA-DR, CD86, CD83 and IL12 (p70) dendritic cells comprisingincubating said DC with activated δγ T cells.

The inventors found that coculture of DC with activated γδ T cellsresulted in the upregulation of HLA-DR, CD86, CD83 surface markers. Thisupregulation is comparable with the increase of said surface markers onDC when using non-activated γδ T cells.

As mentioned above non-activated γδ T cells could stimulate IL12(p40)synthesis in DC. Present invention illustrates that superinduction ofIL-12 (p40) synthesis in DC is possible when γδ T cells and DC arecocultured in presence of a γδ T-cell activator (for instance BrHpp).Surprisingly, the inventors found that γδT cells, stimulated with BrHpp,did elicit IL-12 (p70) production by DC; this in contrast to theinduction of only IL12(p40) when treated with non-preactivated γδ Tcells. Therefore, the inventors defined the differentiated cells ofpresent invention, using activated γδ T cells, as HLA-DR, CD86, CD83 andIL12 (p70) dendritic cells. Also in this method γδ T cells from freshblood, in vitro induced γδ T cells, or cells which can be differentiatedinto γδ T cells can be used.

The inventors point towards the fact that the method according topresent invention for the differentiation and/or maturation of immaturemyeloid dendritic cells (DC) into HLA-DR, CD86, CD83 and IL12 (p70)dendritic cells comprising incubating said DC with activated γδ T cellscan be performed in vitro or in vivo. For example, activated γδ T cellsor γδ T cells supplemented with an activating agent, can be injected inpatients resulting in the in vivo production of HLA-DR, CD86, CD83 andIL12 (p70) dendritic cells. Alternatively, the activating agent can beinjected ‘as such’ resulting in the activation of endogenous γδ T cells.The inventors suggest that these HLA-DR, CD86, CD83 and IL12 (p70)dendritic cells have an even higher capacity, compared to the HLA-DR,CD86, CD83 and IL12 (p40) dendritic cells, to induce efficient Th1-typeand CTL responses.

According to present invention, said activated γδ T cells are producedby treating γδ T cells with an activating agent chosen from microbialproducts or derivatives thereof. Microbial products such asphosphoantigens are produced by Gram-positive and Gram-negative bacteriaand also by some eukaryotic parasites and plants. Mycobacteriumtuberculosis produces four distinct phosphoantigens. These moleculesshare a moiety that is responsible for the potent stimulation of γδ-Tcells seen in tuberculosis patients. The structure of this common coreis 3-formyl-1-butyl-pyrophosphate, a recently described phosphoester.Synthetic analogues of natural phosphoantigens are also known. RecentlyEspinosa et al. have developed a synthetic reagent called BrHpp whosebiological properties on human γδ T cells are optimized compared tothose of 3-formyl-1-butyl-pyrophosphate. As the inventors in presentpatent application showed that δγ-T cells communicate with DC, BrHpp wasused to study the effect of BrHpp-treated γδ-cells. The inventors foundthat this synthetic analogue could also further stimulate the DCmaturation. Therefore, said derivate, bromohydrin pyrophosphate (BrHpp),is used as an example in present invention to further induce DCmaturation. In addition, it is likely that γδ T cells have to beactivated to induce DC maturation in vivo. Therefore, the inventorspropose BrHPP as an adjuvant able to boost Th1 and CTL responses throughits ability to induce DC maturation.

According to present invention, BrHpp may be present in the method ofpresent invention at a concentration between 10 and 1000 nM. When BrHppis present in a too low concentration, no induction is expected. Forexample, BrHpp may be present at a concentration of 200 nM.

Also here, immature myeloid DC, derived from monocytes through cytokinetreatment chosen from IL-4/GM-CSF or IFN-β/IL-3, or functional analoguesthereof, may be used to prepare HLA-DR, CD86, CD83 and IL12 (p70)dendritic cells.

According to present invention, said cytokines may be addedsimultaneously, sequentially or separately with the δγ T cells to themonocytes. In the examples, evidence is shown that said immature myeloidDCs are further maturated by the activated γδ cells interaction.Nevertheless, the inventors do not exclude the possibility that asimilar result may be obtained by first contacting the monocytes withthe γδ T cells prior to the cytokine treatment. In addition, it isobvious for a person skilled in the art that both steps may be performedsimultaneously or that the activating agent, γδ T cells and monocytesare mixed at the same time.

According to the present invention, the cell ratio of the activated δγ Tcells over the monocytes/dendritic cells is between 0.1 and 10. Forexample, the cell ratio of the activated δγ T cells over themonocytes/dendritic cells may be 1:1.

The present invention further contemplates a population of HLA-DR, CD86,CD83, IL12 (p40) dendritic cells and/or a population of HLA-DR, CD86,CD83, IL12 (p70) dendritic cells obtainable by a method according topresent invention.

In present invention experimental evidence is given showing thatnon-activated γδT cells induce the production of IL-12 (p40) in immaturemyeloid DC. In contrast, activated γδT cells induced the production ofIL-12 (p70) by DC, an effect that involved IFN-γ production stimulatedby BrHpp. Therefore, the present invention also relates to a method toproduce IL12 (p40) or IL12 (p70) using a population of HLA-DR, CD86,CD83 and IL12 (p40) dendritic cells or HLA-DR, CD86, CD83 and IL12 (p70)dendritic cells, respectively. This production can be performed in vitroor in vivo.

The relevance of this finding to DC function is further demonstrated inpresent invention by the increased production of IL-5 or IFN-γ byallogenic T cells (CD4⁺ T cells) when stimulated in mixed leucocytereaction LR with DC pre-incubated with nonactivated or activated γδTcells, respectively. Therefore the present invention also provides amethod to produce (further induce) IL-5 by alloreactive T cells using apopulation of HLA-DR, CD86, CD83 and IL12 (p40) dendritic cellsaccording to present invention. The present invention also provides amethod to produce (further induce) IFN-γ by alloreactive T cells using apopulation of HLA-DR, CD86, CD83 and IL12 (p70) dendritic cellsaccording to present invention. This production can be performed invitro or in vivo. With ‘alloreactive T cells’ is meant T cellsspecifically recognizing foreign major histocompatibility molecules.

The present invention describes a method for obtaining a population ofHLA-DR, CD86, CD83 and IL12(p40) dendritic cells, comprising at leastthe following steps:

-   (a) isolating monocytes from a patient,-   (b) incubating said monocytes in the presence of IFN-D/IL-3,    GM-CSF/IL-4 or functional analogues thereof, producing a population    of immature myeloid dendritic cells,-   (c) isolating γδ T cells, and,-   (d) contacting immature myeloid dendritic cells of step (b) with T    cells of step (c), whereby said contact can be performed directly or    indirectly. (claim 21)

There are two main sources of DC precursors: CD34+ stem cells andperipheral blood (PB) monocytes. The main constraints of generating DCfrom stem cells is that the culture time is long and obtaining CD34+cells requires mobilization of the patient. Therefore a preferredembodiment of the present invention is to use monocytes as a DCprecursor. These cells can normally when present in blood differentiateinto DC after a 7 day culture in presence of GM-CSF/IL4 or In presenceof IL3/IFN-β. Different techniques might be used to isolate monocytesfrom the blood as known by the person skilled in the art. With the term“population” is meant HLA-DR, CD86, CD83 and IL12(p40) dendritic cellsas such, a group of HLA-DR, CD86, CD83 and IL12(p40) dendritic cellswhich may be different in other characteristics, or a group of cellscomprising HLA-DR, CD86, CD83 and IL12(p40) dendritic cells. Also onecell is not excluded from this definition. It is important to mentionthat, the inventors do not exclude the fact that monocytes can befurther differentiated and maturated in vivo by injecting the immaturemyeloid dendritic cells and γδ T cells as such into the patient.

The inventors demonstrated that cell to cell contact is not required forthe induction of DC maturation by γδT cells as it was also observed whenthe two cell populations were seeded in transwells. Nevertheless, cellto cell contacts involving membrane-bound molecules could alsoparticipate as the residual production of IL-12 in the presence ofanti-IFN-γ Ab decreased when cells were separated in transwells (datanot shown). According to present invention, the contacting of step maybe performed for 24 hours.

However it is evident for a person skilled in the art that variation onthis incubation time is possible. Present inventors suggest to performthe incubation preferentially between 8 and 48 hours.

The present invention provides a method for obtaining a population ofHLA-DR, CD86, CD83 and IL12(p70) dendritic cells, comprising at leastthe following steps:

-   (a) isolating monocytes from a patient,-   (b) incubating said monocytes in the presence of IFN-β/IL-3,    GM-CSF/IL-4 or functional analogues thereof, producing a population    of immature myeloid dendritic cells,-   (c) isolating γδ T cells,-   (d) culturing T cells of step (c) with microbial products or    derivatives thereof as defined above, thereby activating said γδ T    cells, and,-   (e) contacting dendritic cells of step (b) with T cells of step (d),    whereby said contact is performed directly or indirectly.

According to present invention, the contacting of step may be performedfor 24 hours.

However it is evident for a person skilled in the art that variation onthis incubation time is possible. Present inventors suggest to performthe incubation preferentially between 8 and 48 hours.

According to present invention HLA-DR, CD86, CD83 and IL12(p40)dendritic cells may further be treated to produce antigen presentingdendritic cells. Consequently the method as described by the presentinvention comprises at least the following steps:

-   (a) isolating monocytes from a patient,-   (b) incubating said monocytes in the presence of IFN-β/IL-3,    GM-CSF/IL-4 or functional analogues thereof, producing a population    of immature myeloid dendritic cells,-   (c) isolating γδ T cells,-   (d) contacting dendritic cells of step (b) with T cells of step (c),    whereby the contact is performed directly or indirectly, and,-   (e) presenting a peptide on the surface of said dendritic cells.

According to present invention HLA-DR, CD86, CD83 and IL12(p70)dendritic cells may further be treated to produce antigen presentingdendritic cells. Consequently the method as described by the presentinvention comprises at least the following steps:

-   (a) isolating monocytes from a patient,-   (b) incubating said monocytes in the presence of IFN-β/IL-3,    GM-CSF/IL-4 or functional analogues thereof, producing a population    of immature myeloid dendritic cells,-   (c) isolating of γδ T cells,-   (d) culturing T cells of step (c) with microbial products or    derivatives thereof, thereby activating said γδ T cells,-   (e) contacting dendritic cells of step (b) with T cells of step (d),    whereby said contact is performed directly or indirectly, and,-   (f) presenting a peptide on the surface of said dendritic cells.

Depending on the specific treatment as described below, antigens arespecific molecules present on cells selected from the group consistingof a cancer cell, a bacteria, a parasitically infected cell and avirally infected cell. These antigens can be large molecules which areprocessed by the DC to load MHC molecules, or can be smaller molecules(i.e. peptides) which are immediately loaded onto the MHC molecules.Several approaches have been used to arm DC with target antigen for usein clinical trials. Methods used to approach this step of antigenloading are reviewed by Fong and Engleman ³⁶. Inventors also point outthat, HLA-DR, CD86, CD83 and IL12(p70) dendritic cells can be producedin vivo by injecting γδ T cells in combination with an antigen into thepatient.

The capacity of presenting a peptide on the surface of said dendriticcells according to present invention can for example be achieved bycontacting said dendritic cell with at least part of an antigendifferentially expressed on a cell. This cell can be a cell selectedfrom the group consisting of a cancer cell, a bacterial cell, aparasitically infected cell and a virally infected cell. Antigens aredelivered from these to the DC resulting in the activation of the DCs.

Alternatively, the capacity of presenting a peptide on the surface ofsaid dendritic cells can be achieved by pulsing said dendritic cellswith antigenic proteins, by loading said dendritic cells with antigenicpeptides or can be achieved by transforming/transducing said dendriticcells by nucleic acid molecules coding for at least part of saidantigen. With “pulsing” is meant that DC are activated by these antigensand enter into the MHC class II and/or MHC class I processing pathway.Transformation of DC can be achieved using electric pulses, liposomes orother techniques as known by the person skilled in the art. Viralvectors allow the transduction of cells. With viral vectors alsoretroviral, adenoviral and adeno-associated vectors are meant.

Transformation/transduction of the cells allows introduction of DNAencoding the antigen and when appropriate expression signals are presentsaid antigen is made in the cell and brought through the endogenousmechanisms to the surface of the transformed/transduced dendritic cell.As a result of this an antigen-presenting HLA-DR, CD86, CD83 andIL12(p40) dendritic cell or an HLA-DR, CD86, CD83 and IL12(p70)dendritic cells is made.

Alternatively, the capacity of presenting a peptide on the surface ofsaid dendritic cells is achieved by fusing said dendritic cell withcells carrying specific antigens. The production of dendritic-likecell/tumor cell hybrids and hybridomas for inducing anti-tumor responsehave been described in WO96/30030. This document provides dendritic-likecell/tumor cell hybridomas and pluralities of dendritic-like cell/tumorcell hybrids that confer tumor resistance in vivo. The hybrids andhybridomas are generated by the fusion of tumor cells withdendritic-like cells. For instance, immortal tumor cells from anautologous tumor cell line can be fused with autologous or HLA-matchedallogeneic dendritic-like cells. Autologous tumor cell lines can bederived from primary tumors and from their metastases. Alternatively,immortal dendritic-like cells from an autologous or allogeneicHLA-matched dendritic-like cell line can be fused with autologous tumorcells. Autologous dendritic-like cell lines can be prepared from varioussources such as peripheral blood and bone marrow. Dendritic-likecell/tumor cell hybridomas and pluralities of hybrids can be directlyinfused for active immunization of cancer patients against theirresidual tumor cells. The hybridomas and hybrids can also be used forthe in vitro activation of autologous immune cells before theirreinfusion into the patient for passive immunization against the tumorcells.

The present invention also proposes a method to provide an activatedpopulation of T cells using antigen-presenting HLA-DR, CD86, CD83 andIL12 (p40) dendritic cells obtainable by a method as described abovecomprising at least the following steps:

-   (a) isolating monocytes from a patient,-   (b) incubating said monocytes in the presence of IFN-β/IL-3,    GM-CSF/IL-4 or functional analogues thereof, producing a population    of immature myeloid dendritic cells,-   (c) isolating γδ T cells,-   (d) contacting dendritic cells of step (b) with T cells of step (c),    whereby said contact is performed directly or indirectly,-   (e) presenting a peptide on the surface of said dendritic cells,    thereby providing a population of antigen presenting dendritic    cells; and,-   (f) activating a population of T cells with said population of    antigen presenting dendritic cells of step (e).

The inventors point towards the fact that the activated population of Tcells may represent both CD4Th1 and/or CD8 cytotoxic T cells (CTL).

A method for activating a T cell using HLA-DR, CD86, CD83 and IL12 (p70)dendritic cells obtainable by a method as described in the previousclaims comprising at least the following steps:

-   (a) isolating monocytes from a patient,-   (b) incubating said monocytes in the presence of IFN-β/IL-3,    GM-CSF/IL-4 or functional analogues thereof, producing a population    of immature myeloid dendritic cells,-   (c) isolating γδ T cells,-   (d) culturing T cells of step (c) with microbial products or    derivatives thereof, thereby activating said γδ T cells,-   (e) contacting dendritic cells of step (b) with T cells of step (d),    whereby said contact is performed directly or indirectly,-   (f) presenting a peptide on the surface of said dendritic cells,    thereby providing a population of antigen presenting dendritic    cells; and,-   (g) activating a population of T cells with said population of    antigen presenting dendritic cells of step (f).

An activated T cell being a T cell (CD3+ cell) proliferating and/orsecreting cytokines (IL-2, IL-4, IL-5, IFN-γ, etc.) and/or expressingactivation markers (CD25, CD69, HLA-DR, CD40L, etc.). If necessary, anactivated T cell can always be separated from the antigen presentingdendritic cell by cell sorting.

In preferred methods according to present invention said T cell is a Thelper cell.

The invention also refers to a method as described above wherein thesteps of producing a population of cells as described above such asHLA-DR, CD86, CD83 and IL12 (p40) dendritic cells, HLA-DR, CD86, CD83and IL12(p70) dendritic cells, antigen presenting HLA-DR, CD86, CD83 andIL12(p40) dendritic cells, antigen presenting HLA-DR, CD86, CD83 andIL12(p70) dendritic cells and/or activated T cells using antigenpresenting HLA-DR, CD86, CD83 and IL12(p40) dendritic cells or antigenpresenting HLA-DR, CD86, CD83 and IL12(p70) dendritic cells are carriedout in vitro and/or in vivo.

The present invention also provides a population of antigen-presentingHLA-DR, CD86, CD83 and IL12(p40) dendritic cells, a population ofantigen-presenting HLA-DR, CD86, CD83 and IL12(p70) dendritic cells or apopulation of activated T cell obtainable by a method as describedabove.

The present invention also relates to a composition for use as amedicament or a cell based product intended for clinical use comprisingat least one of the following components according to present invention:

-   -   a population of HLA-DR, CD86, CD83 and IL12(p40) dendritic        cells,    -   a population of HLA-DR, CD86, CD83 and IL12(p70) dendritic        cells,    -   a population of antigen-presenting HLA-DR, CD86, CD83, IL        12(p40) dendritic cells,    -   a population of antigen-presenting HLA-DR, CD86, CD83, IL12(p70)        dendritic cells,    -   a population of antigen-presenting HLA-DR, CD86, CD83, IL12(p40)        dendritic cells mixed with antigen,    -   a population of antigen-presenting HLA-DR, CD86, CD83, IL12(p70)        dendritic cells mixed with antigen,    -   a population of activated T cells obtainable using        antigen-presenting HLA-DR, CD86, CD83, IL12(p40) dendritic        cells, or,    -   a population of activated T cells obtainable using        antigen-presenting HLA-DR, CD86, CD83, IL12(p70) dendritic        cells.

Cell based products are not yet considered as medicament and could beconsidered as transfusion products in the future.

Therefore the present inventors concluded that upon using this specificmethod a new type of DC can be formed which is more mature thanpreviously described myeloid DC and which have a superior character ininducing T cell expression. Increased cytokine expression results in amore rapid and efficient stimulation of the immune system, and thereforewill be more efficient in eliminating foreign infectious material in apatient.

As IL-12 (p70) is the bioactive form of IL12 (p40) the Inventors assumethat HLA-DR, CD86, CD83 and IL12(p70) dendritic cells will have a moreimportant effect on the induction of the Th1 and CTL response comparedto the HLA-DR, CD86, CD83 and IL12(p70) dendritic cells.

Monocyte-derived DC primed with tumor antigens are now clinically usedin several protocols to induce specific antitumor immunity. Both Th1 andTh2 effector mechanisms have been shown to collaborate with each otherin directing an effective antitumor activity. Because of their abilityto induce both Th1 and Th2 type responses, the inventors suggest thatHLA-DR, CD86, CD83 and IL12 (p40) dendritic cells and HLA-DR, CD86, CD83and IL12 (p70) dendritic cells might be appropriate to induce efficienttumor immunity.

A composition according to the invention may be supplemented with atleast one additional cytokine. According to the present invention, saidcytokine is may for instance be chosen from a group comprising IFN-α,IFN-β, IL-3 and IL-12. IL-12 and IFN-α are pivotal cytokines for Th1differentiation and generation of cytotoxic T cells endowed with potentanti-tumor effects.

The invention implies the use of a compound according to presentinvention for the preparation of a medicament. Specifically, thesecompounds can be used for the preparation of a medicament for thetreatment and/or prevention of a disease whereby stimulation of the Th1and/or CTL response is needed comprising a composition as describedabove. These diseases can be chosen from the group comprising cancer,infections and autoimmune diseases. Investigations showed that theimmunologic and clinical effects of antigen-loaded dendritic cellsadministered as a therapeutic vaccine to patients with cancer. AlthoughDC-based vaccination methods are cumbersome, promising results fromclinical trials in patients with malignant lymphoma, melanoma, andprostate cancer suggest that immunotherapeutic strategies that takeadvantage of the antigen-presenting properties of dendritic cells mayultimately prove both efficacious and widely applicable to human tumors.Also the role of DC in initiating or priming immune responses to viraland bacterial antigens in vivo is well established. It has beendemonstrated that human DC, but not monocytes or B cells, can sensitisenaïve T cells to soluble protein antigens, enabling the generation ofantigen-specific CD4+ helper and CD8+ CTL lines in vitro. CD8+ cytotoxicT lymphocytes (CTL) have been demonstrated to recognize and kill cancercells in various tumor models. The ability of DC to prime T cellscapable of recognizing and killing tumor cells in an antigen-specificfashion has been demonstrated in various animal models. Moreover,DC-based immunization can lead to immunologic memory with protectionagainst subsequent tumor challenges. Fong et al (1997) illustrated thatimmunizing with self proteins could protect animals against autoimmunereactions.

The present invention also relates to the use of microbial products(such as phosphoester, BrHpp, derivatives or combinations thereof) forthe preparation of a medicament for the treatment and/or prevention of adisease whereby stimulation of the Th1 and/or CTL response is needed.Also here, said disease can be chosen from the group consisting ofcancer, infections and autoimmune diseases. The inventors point herebyto the fact that BrHpp can be used as an adjuvant to elicit DCmaturation in vivo.

The present invention also relates to the pharmaceutical compositioncomprising at least one of the components according to the invention andoptionally a pharmaceutical acceptable carrier, diluent or excipient.Pharmaceutically acceptable carriers include any carrier that does notitself induce the production of antibodies harmful to the individualreceiving the composition. Suitable carriers are typically large, slowlymetabolizing macromolecules such as proteins, polysaccharides,polylactic acids, polyglycolic acids, polymeric amino acids, amino acidcopolymers; and inactive virus particles. Such carriers are well knownto those of ordinary skill in the art. A “vaccine” is an immunogeniccomposition capable of eliciting protection against infections, whetherpartial or complete. A vaccine may also be useful for treatment of anindividual, in which case it is called a therapeutic vaccine. Saidvaccine compositions may include prophylactic as well as therapeuticvaccine compositions. The term “therapeutic” refers to be capacity ofeliminating or preventing invasive cells.

The present invention also relates to a method of killing a target cellcomprising contacting said target cell with a composition. This killingcan be performed in vitro or in vivo. Said target cell may for instancebe selected from the group consisting of a cancer cell, a bacterialcell, a parasitically infected cell or a virally-infected cell.

The present invention also provides an in vitro screening method using apopulation of HLA-DR, CD86, CD83 and IL12(p40) dendritic cells, apopulation of HLA-DR, CD86, CD83 and IL12(p70) dendritic cells, apopulation of antigen-presenting HLA-DR, CD86, CD83 and IL12(p40)dendritic cells, a population of antigen-presenting HLA-DR, CD86, CD83and IL12(p70) dendritic cells or a population of activated T cellsobtainable by a method as described above. By their potentimmunostimulatory properties, DC loaded with tumor or bacterial Ag couldbe used to activate T cells against unknown poorly immunigenic Ag andthus help to discover them.

According to present invention, a population of HLA-DR, CD86, CD83 andIL12(p40) dendritic cells, a population of HLA-DR, CD86, CD83 andIL12(p70) dendritic cells, a population of antigen-presenting HLA-DR,CD86, CD83 and IL12(p40) dendritic cells, a population ofantigen-presenting HLA-DR, CD86, CD83 and IL12(p70) dendritic cells or apopulation of activated T cell obtainable by a method according to theinvention can be used for the preparation of in vitro screening tests.

According to present invention, a method for detecting T cell mediatedactivity of a target antigenic peptide, comprising at least thefollowing steps:

-   (a) providing a population of antigen-presenting HLA-DR, CD86, CD83    and IL12(p40) dendritic cells or a population of antigen-presenting    HLA-DR, CD86, CD83 and IL12(p70) dendritic cells obtainable    according to a method as described above,-   (b) contacting a T cell with said dendritic cell, thereby providing    an activated T cell,-   (c) contacting a target cell with said activated T cell, and,-   (d) monitoring the effect of said activated T cell on said target    cell, thereby detecting anti-target activity.

The present invention also describes a kit for detecting T cell mediatedactivity of a target antigenic peptide, comprising at least one of thefollowing components according to present invention:

-   -   a population of HLA-DR, CD86, CD83 and IL12(p40) dendritic        cells,    -   a population of HLA-DR, CD86, CD83 and IL12(p70) dendritic        cells,    -   a population of antigen-presenting HLA-DR, CD86, CD83, IL12(p40)        dendritic cells,    -   a population of antigen-presenting HLA-DR, CD86, CD83, IL12(p70)        dendritic cells,    -   a population of antigen-presenting HLA-DR, CD86, CD83, IL12(p40)        dendritic cells mixed with antigen,    -   a population of antigen-presenting HLA-DR, CD86, CD83, IL12(p70)        dendritic cells mixed with antigen,    -   a population of activated T cells obtainable using        antigen-presenting HLA-DR, CD86, CD83, IL12(p40) dendritic        cells, or,    -   a population of activated T cells obtainable using        antigen-presenting HLA-DR, CD86, CD83, IL12(p70) dendritic        cells.

It has been shown that freezing population of said cells did not alterthe functional properties of these cells. Also other methods for storageas known by the skilled person in the art can be applied to preservethese cells.

All methods, uses and kits described in present Invention for thedetection of T cell mediated activity also relate to the use of apopulation of HLA-DR, CD86, CD83 and IL12 (p40) dendritic cells and/orHLA-DR, CD86, CD83 and IL12 (p70) dendritic cells as reagent for thepurpose of following the immune response in patients who got eitherDC-vaccines or other vaccines. T cells might be isolated from patientsand tested using antigen-presenting HLA-DR, CD86, CD83 and IL12 (p40)dendritic cells or antigen-presenting HLA-DR, CD86, CD83 and IL12 (p70)dendritic cells to analyse if the immunologic response in the patienthas been activated. For this purpose PBMC (periferal blood mononuclearcells) or purified T cells might be used. This test system allows theevaluation of any therapy against infections, cancer or auto-immunediseases.

The present invention suggests the use of a composition according to theinvention as a vaccine adjuvant and the vaccine adjuvant as suchcomprising a composition according to the invention.

As pointed out before, compositions according to present inventionand/or microbial products, such as BrHpp, can be used as a vaccineadjuvant for the stimulation of DC maturation in vivo.

According to present invention a vaccine comprising the composition asdescribed by the invention can be used to immunize humans or animalsagainst different diseases (adjuvant). Vaccination of patients hasalready been illustrated and found to be efficacious usingpeptide-pulsed IL4/GM-CSF DC in cancer patients (Toungouz et al. 1999).

In particular, the present invention describes a method for immunizinghumans or animals against a disease comprising administering a vaccinecomprising an adjuvant as described above.

In terms of vaccination against infectious diseases, BrHPP mightespecially be of interest in the newborn in which DC are deficient. Theinventors propose that BrHPP might allow to enhance the function ofneonatal DC and therefore when incorporated in vaccine formulationsmight allow in combination with antigens to induce efficient immunityagainst pathogens (i.e. malaria, tuberculosis, HIV) Therefore, thepresent invention also relates to a method of immunizing newborns.

The present invention also relates to a method of treatment of cancer,infections and autoimmune diseases comprising the use of at least one ofthe following compositions according to the invention.

In the design and conduct of above described applications, importantconsiderations include methods for introducing the antigen into MHCclass 1 and 11 processing pathways, methods for isolating and activatingdendritic cells, route of administration and antigen selection. Becausethe cell therapy as presented in the present invention needs a specificrecognition of the target cell, it is important that indeed the choiceof antigen is well considered. Therefore the present invention suggeststhat the antigen is a tumor specific antigen, an infectious specificantigen or a self-protein when applied in the treatment of cancer,infections (viral, bacterial, parasitical) or autoimmune diseases. Inaddition, it is important that the compositions are administered to aperson in need of treatment in a therapeutically effective amount.Example of antigens that might be considered as tumor antigens aredescribed by Fong and Engleman 2000.

According to the present invention said viral disease is selected fromthe group of HIV, human Papilloma virus, Ebstein Barr virus andCytomegalovirus.

According to the present invention said autoimmune disease is selectedfrom the group consisting of multiple sclerosis myasthenia gravis,juvenile chronic arthritis, chronic arthritis, LED, atopic dermatitisand juvenile diabetes. Inventors suggest that probably all autoimmunediseases may be treated or prevented by a method as described by theinvention.

According to the present invention, said compositions can be injectedinto patients using different ways. Injection may for instance becarried out intravenously, intra-lymphoidal or intratumoral,nevertheless, other routes can be used such as subcutaneous injections.It is interesting to mention that in addition to expressing therequisite MHC and costimulatory molecules to prime T cells, the DC cellsexpress appropriate adhesion molecules and chemokine receptors toattract the DC to secondary lymphoid organs for priming. In thisrespect, inefficient priming could be circumvented by injecting DCdirectly to secundary lympoid organs through intralymphatic orintranodal injection. The present study gives evidence that especiallyin cancer treatment intra-tumoral injections will result in moreefficient elimination of the tumor. The inventors showed in EP00870273.0that monocyte-derived IL-3/IFN-β DC are able to trigger apoptosis intumor cells which is relevant to their therapeutic use as anti-tumorvaccines. Indeed, recent reports demonstrated that human IL-4/GM-CSF DCcan process apoptotic cells and cross-present the derived antigens in aMHC-class I restricted fashion, resulting in the induction of efficientcytotoxic T cell responses. Therefore present inventors suggest thatantigen-presenting HLA-DR, CD86, CD83 and IL12 (p40) dendritic cellsand/or antigen-presenting HLA-DR, CD86, CD83 and IL12 (p70) dendriticcells which are directly injected into tumors may first induce apoptosisof cancer cells, and finally migrate in the lymph nodes where theyinduce tumor-specific T-cell responses.

These compositions may, for example, be administered parentally orintravenously. The compositions according to the invention forparenteral administration can be, in particular, sterile solutions,aqueous or non-aqueous, suspensions or emulsions, As a pharmaceuticallyacceptable solution or vehicle propylene glycol, polyethylene glycol,injectable organic esters, for example ethyl oleate, or cyclodextrinsmay be employed. These compositions can also comprise wetting,emulsifying and/or dispersing agents.

The sterilisation may be carried out in several ways, for example, usingbacteriological filter, by incorporating sterilising agents in thecomposition or by irradiation. They may also be prepared in the form ofsterile solid compositions which may be dissolved at the time of use insterile water or any other sterile injectable medium.

The present invention can also comprise adjuvants which are well knownto a person skilled in the art (vitamin C, antioxidant agents, etc.)capable of being used in synergy with the compounds according to theinvention in order to improve and prolong the treatments of canceroustumors.

The invention also relates to a composition comprising a compositionaccording to present invention and another compound as a combinedpreparation for simultaneous, separate or sequential use for treatingcancer, infections and autoimmune diseases.

The present invention also relates to a method for the preparation of acomposition as described by present invention comprising followingsteps:

-   (a) isolating monocytes from a patient,-   (b) incubating said monocytes in the presence of IFN-β/IL-3,    GM-CSF/IL-4 or functional analogues thereof in clinical grade    conditions, producing a population of immature myeloid dendritic    cells,-   (c) isolation of γδ T cells, and,-   (d) contacting immature myeloid dendritic cells of step (b) with T    cells of step (c), in clinical grade conditions,-   (e) presenting an antigen on the surface of said dendritic cells in    clinical grade conditions, thereby providing a population of antigen    presenting dendritic cells; and,-   (f) activating a population of T cells with said population of    antigen presenting dendritic cells. As described above each of these    steps can be performed in vitro and/or in vivo.

Recently improvements were made for the production of DC inclinical-grade conditions. The present inventors described in Toungouzet al 1999³⁸ that the development of closed systems, avoidance ofexogenous proteins and respect of standard operating procedures (SOP) isneeded to be able to guarantee predefined specifications of the cellularproduct. In these documents a good manufacturing practice(GMP)-simplified procedure of IL4/GM-CSF DC generation fromleukapheresis products in a closed system, using synthetic culture mediadevoid of non-human protein is described. In analogy to this method,clinical grade HLA-DR, CD86, CD83 and IL12 (p40) dendritic cells orHLA-DR, CD86, CD83 and IL12 (p70) dendritic cells can be prepared.

Unless other wise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. The materials, methods, and examples are illustrative only andnot intend to be limiting. Other features and advantages of theinvention will be apparent from the following drawings, detaileddescription, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: γδ T cells induce the upregulation of HLA-DR, CD86 and CD83expression on monocyte-derived DC. Monocyte-derived DC were eithercultured in medium alone, in the presence of BrHpp (100 nM), or in thepresence of γδ T cells which were prestimulated or not with BrHpp. DCand γδ T cells were also cocultured in transwells. DC cell surfacemarkers were analyzed after overnight coculture using flow cytometry.One representative experiment out of 6 is shown.

FIG. 2: Role of TNF-α in the upregulation of DC surface moleculesinduced by γδ T cells. Monocyte-derived DC were cocultured withBrHpp-activated γδ T cells in the presence of neutralizing anti-TNF-α(20 ug/ml), anti-IFN-γ (15 ug/ml) mAb or both. After overnight culture,cell surface markers were assessed by flow cytometry. One representativeexperiment out of 5 is shown.

FIG. 3: γδ T cells induce IL-12 production by DC. Monocyte-derived DCwere cocultured alone or in the presence of BrHpp only or coculturedwith unstimulated or BrHpp-activated γδ T in absence or presence ofeither anti-TNF-α, anti-IFN-γ neutralizing mAbs or both. After 24 h ofculture, supernatants were assayed for IL12-p40 and p70 levels by ELISA.Results were expressed as mean±SEM of 6 independent experiments.

-   * p<0.05 compared to DC cultured in medium alone or containing BrHpp-   ** p<0.05 compared to DC stimulated with BrHPP-activated γδ T cells    in the absence of mAb.

FIG. 4: DC were either cultured alone, or in the presence ofunstimulated or BrHpp-stimulated γδ T cells, thereafter irradiated (6000Rads) and finally added to allogenic CD4⁺ T cells. After 5 days,supernatants were assayed by ELISA for IFN-γ and IL-5 levels. Data areshown as mean±SEM of 5 independent experiments. * p<0.05 as comparedwith DC that were not precultured with γδ T cells.

Table 1: TNF-α and IFN-γ production by γδ T cells.

γδ T cells (7.5 10⁵ cells/500 μl) were either cultured in medium aloneor stimulated with BrHpp (200 nM). After 48 h, culture supernatants wereassayed by ELISA for TNF-α and IFN-γ levels. Data are shown as mean±SEMof 13 independent experiments.

-   ¹ p<0.003 as compared to medium alone (without BrHpp)    Table 2: Phenotypic Changes of DC upon Coculture with γδ T cells.

DC cultures were cultured for 24 alone, in the presence of BrHpp (200nM) only, or with γδ T cells activated by BrHpp as described inexample 1. Neutralizing anti-TNF or anti-IFNγ Ab was added at aconcentration of 20 and 15 μg/ml, respectively. The expression ofHLA-DR, CD86, and CD83 on DC was measured by flow cytometry andexpressed as means+/−SEM of mean fluorescence intensity in fiveindependent experiments on different healty donors.

-   ¹ p<0.05 compared to DC cultured alone or with BrHpp only.-   ² p<0.05 compared to DC cultured with activated γδ T cells in    absence of mAb.

MODES FOR CARRYING OUT THE INVENTION EXAMPLE 1 Material and Methods

Reagents and Medium. The phosphoantigen bromohydrin pyrophosphate(BrHpp) was kindly provided by Innate Pharma (Marseille, France).Culture medium consisted of RPMI-1640 (Life-Technologies, Paisley,Scotland) supplemented with 50 μM mercaptoethanol, 20 μg/ml gentamycin,2 mM L-glutamine, 1% nonessential amino acids (Life Technologies) andFBS-10% (Perbio, Aalst, Belgium).

Purification of γδ T cells and DC generation. Peripheral bloodmononuclear cells (PBMC) from healthy volunteers were isolated bydensity centrifugation of heparinized blood on Lymphoprep (Nycomed,Oslo, Norway), washed with HBSS, resuspended in culture medium andallowed to adhere In culture flasks for 2 h at 37° C. Non-adherent cellswere removed and adherent monocytes were cultured during 6 days inpresence of 500 U/ml granulocyte macrophage colony-stimulating factor(GM-CSF) (Leucomax, Schering-Plough Kenilworth, N.J.) and 800 U/ml ofIL-4 (Cellgenix, Freiburg, Germany). The resulting cell preparationroutinely contained >90% DC as assessed by morphology and FACS analysis.

For γδ T cell isolation, cells expressing γ-δ receptors on theirmembrane were positively selected using immunomagnetic depletion(Miltenyi Sanvertech, Belgium). Briefly, non-adherent PBMC containing 2to 5% of γδ T cells were incubated with biotin-conjugated anti-γδ T cellreceptor (TCR) antibodies for 15 min at 4° C., washed three times, andthen incubated with immunomagnetic beads coated with streptavidin.Positively selected populations routinely contained more than 90% viableγδ T cells as assessed by flow cytometry. Those cells were positive forCD3 and γδ TCR and expressed neither CD25 not CD40L.

Cell culture conditions. γδ T cells (7.5 10⁵ cells/500 μl) were culturedfor 24 h in flat-bottomed 24-well plates in culture medium supplementedor not with BrHpp (200 nM). Autologous DC (10⁶ cells/500 μl) were addedto γδ T cell cultures for another 24 h and analyzed for the expressionof surface markers and for their ability to release cytokines. Inparallel, DC (10⁶ cells/ml) were cultured for 24 h in 24-well plateseither in medium alone, or in presence of BrHpp (100 nM). In someexperiments, anti-TNF-α (20 μg/ml) or anti-IFN-γ (15 μg/ml) neutralizingmonoclonal antibody (mAb) or their isotypic control used at similarconcentration (Biosource Fleurus, Belgium) were added to DC-γδ T cellcocultures. In parallel, DC and γδ T cells were cocultured in atranswell culture system (Costar, Belgium).

Determination of cytokine levels. TNF-α, IL-12 p-40 and IFN-γ levels inculture supernatants were determined by ELISA kits from Biosource. IL-12p70 levels were measured using the Endogen Elisa kit (Endogen, Belgium).

Immunophenotyping by flow cytometry. Monocyte-derived DC were stainedusing PE-labeled specific mAb HLA-DR, CD86, CD83 (Beckton-Dickinson,Mountain view, CA). FITC-conjugated anti-TCR γδ mAb (Becton-Dickinson,San Jose, Calif.) was used to assess γδ T cell purity and to excludethem in flow cytometry analysis of DC in DC-γδ T cell cocultures.Briefly, 5×10⁵ cells were incubated with the relevant mAbs or theirisotype-matched controls for 20 min at 4° C., washed and fluorescenceintensity was analyzed using a FACS-Calibur (Becton-Dickinson).

Mixed leucocyte reactions (MLR). 2×.10⁵ CD4⁺ T cells purified from thePBMC of healthy donors using Miltenyi beads were seeded in mixedcultures with irradiated (6000 rads) allogenic DC (2×.10⁴ DC/well). DCwere either unstimulated or pre-activated by coculture for 24 h withautologous γδ T cells in presence of 200 nM BrHpp. After 5 days, mixedleucocyte reaction (MLR) supernatants were assayed for IFN-γ and IL-5 byELISA.

Statistical analysis. Statistical analysis was performed using a nonparametric Wilcoxon test.

EXAMPLE 2 Human γδ T Cells Induce Upregulation of HLA-DR, CD86 and CD83Expression on Monocyte-Derived Dendritic Cells: Role of TNF-α

In a first set of experiments, the inventors analyzed by flow cytometryHLA-DR, CD86 and CD83 expression on dendritic cells derived from PBMCcultured in IL-4 and GM-CSF. As shown in FIG. 1 and table 2, cocultureof DC with γδ T cells resulted in the upregulation of these surfacemarkers, indicating that DC undergo some degree of maturation under theinfluence of γδT cells. Preactivation of γδT cells with BrHpp did notresult in a further increase of this effect. Cell to cell contact wasnot required for the induction of DC maturation by γδT cells as it wasalso observed when the two cell populations were seeded in transwells(FIG. 1 and table 2). As γδT cells are known to secrete TNF-α, theinventors considered the possibility that this cytokine was responsiblefor the action of γδT cells on DC. Indeed, the inventors found that γδTcells directly isolated from blood produced significant amounts ofTNF-α, even in absence of in vitro stimulation (table 1). This In vitroproduction of TNF-α by purified γδ T cells could be related to theisolation procedure. BrHpp further increased this basal production ofTNF-α and also induced IFN-γ secretion by γδ T cells (table 1). Additionof a neutralizing anti-TNF-α mAb to the DC-γδT cells cocultures clearlyreduced the upregulation of HLA-DR, CD86 and CD83 whereas anti-IFN-γ mAbfailed to do so (FIG. 2 and table 2). These data establish a key rolefor TNF-α in the maturation of DC elicited by γδ T cells.

EXAMPLE 3 γδ T Cells Stimulate IL-12 Production by Dendritic Cells:Involvement of IFN-γ

The capacity of DC to induce efficient Th1-type and CTL responses islinked at least in part to their synthesis of IL-12. The inventorstherefore investigated in coculture experiments the impact of γδ T cellson the synthesis by DC of IL-12 (p40) and IL-12 (p70), the bioactiveheterodimeric form of the cytokine. Freshly isolated γδT cells inducedthe production of IL-12 (p40) even in the absence of stimulation byBrHpp. In the presence of BrHpp, a 3-fold increase in IL-12 (p40) levelswas observed, and the induction of IL-12 (p70) synthesis was alsodetected in this setting (FIG. 3). As BrHpp had no effect on DC culturedin absence of γδ T cells (FIG. 3), the inventors concluded thatactivation of γδ T cells by BrHpp was responsible for the induction ofIL-12 synthesis when γδ T cells and DC were cocultured in the presenceof BrHpp. Whereas freshly isolated γδ T cells did not elicit IL-12 (p70)production by DC, they did so when stimulated by BrHpp (FIG. 3). Theaddition of a neutralizing anti-IFN-γ mAb significantly inhibited theinduction of both IL-12 (p40) and IL-12 (p70) synthesis, whereas ananti-TNF-α mAb had no effect (FIG. 3). The inventors conclude from theseexperiments that γδ T cells induce IL-12 production by DC and that thiseffect partially involves IFN-γ. Cell to cell contacts involvingmembrane-bound molecules could also participate as the residualproduction of IL-12 in the presence of anti-IFN-γ Ab decreased whencells were separated in transwells (data not shown). CD40-CD40Linteractions were not responsible for the residual production of IL-12in the presence of anti-IFNγ mAb, as CD40L was not found by flowcytometry at the surface of the δγ T cells even after BrHpp stimulation,and the addition of a blocking anti-CD-40L mAb did not modify IL-12production in DC-γδ T cell cocultures (data not shown).

EXAMPLE 4 Increased Allostimulatory Capacity of DC Cultured in Presenceof Activated γδ T Cells

In order to determine the relevance of the effects of γδ T cells on DC,DC pre-cultured in presence of unstimulated or BrHpp-activated γδ Tcells were irradiated and then seeded as stimulators in mixed leucocytereaction (MLR) with allogenic CD4⁺ T cells for 5 days. Compared tocontrol DC, DC pre-cultured with unstimulated γδ T cells induced theproduction of increased amounts of IL-5 but not IFN-γ by alloreactive Tcells (FIG. 4). On the other hand, DC pre-cultured with BrHpp-activatedγδT cells induced significantly higher levels of IFN-γ in MLR whereasIL-5 levels were not significantly modified (FIG. 4). These data mightbe related to the different levels of IL-12 produced by DC pre-culturedin presence of unstimulated or BrHPP-activated γδT cells. Indeed, thelow levels of IL-12 produced by the former should favor Th2-typeresponses whereas the increased levels produced by the latter shouldpromote Th1-type responses (17). In each case, the immunogenic potentialof DC increased following the interactions with γδ T cells, probably asa consequence of the upregulation of key surface molecules reported hereabove.

EXAMPLE 5 Discussion

The findings reported by the inventors establish a new link betweeninnate immunity and the induction of acquired T cell responses. Indeed,γδ T cells are rapidly activated in the course of several infectionswhere they provide a primary protection (18, 19). The activation of DCthat they simultaneously induce might be critical for the development ofefficient CD4⁺ and CD8⁺ T cell responses. Furthermore, the observationmade by the inventors that synthetic ligands of the Vγ9/Vδ2 TCR induceDC activation suggest that these agents should be considered aspotential vaccine adjuvants. They might be of special interest for earlylife immunization against intracellular pathogens as the inventorsrecently demonstrated that neonatal DC display a defect in IL-12 (p70)synthesis which can be corrected by IFN-γ (20). Along the same line, theinventors currently investigate the possibility that the efficient Th1responses induced in human newborns by the Bacillus Calmette-Guerin(BCG) vaccine (21) are related at least in part to activation of γδ Tcells by mycobacterial phosphoantigens.

EXAMPLE 6 Summary of the Invention

γδ T cells are known to be involved in the innate immune defensesagainst infectious micro-organisms. In this patent application, theinventors considered that γδT cells could also influence acquiredimmunity by interacting with dendritic cells (DC) in the early phase ofthe immune response. To investigate this hypothesis, γδ T cells isolatedfrom peripheral blood of healthy volunteers were cocultured withautologous monocyte-derived dendritic cells which were subsequentlyanalyzed for their expression of key surface molecules and for theirproduction of IL-12. First, the inventors found that γδT cells inducedthe upregulation of HLA-DR, CD86 and CD83 on DC. This effect did notrequire cell to cell contact and could be blocked by a neutralizinganti-TNF antibody. In the same system, γδT cells induced the productionof IL-12 (p40) but not IL-12 (p70) by DC. The inventors then assessedthe consequence of γδT cell activation by the synthetic phosphoantigenbromohydrin pyrophosphate (BrHpp). γδT cells activated by the syntheticphosphoantigen bromohydrin pyrophosphate (BrHpp) induced the productionof IL-12 (p40) and IL-12 (p70) by DC, an effect that involved IFN-γproduction. The relevance of this finding to DC function wasdemonstrated by the increased production of IFN-γ by alloreactive Tcells when stimulated in MLR with DC pre-incubated with activated γδTcells. The inventors conclude that γδT cell activation might result inDC maturation and thereby in enhanced αβ T cell responses.

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21. Marchant, A., Goetghebuer, T., Ota, M. O., Wolfe, I., Ceesay, S. J.,De Groote, D., Corrah, T., Bennett, S., Wheeler, J., Huygen, K., Aaby,P., McAdam, K. P. and Newport, M. J., Newborns develop a Th1-type immuneresponse to Mycobacterium bovis bacillus Calmette-Guérin vaccination. J.Immunol. 1999. 163: 2249-2255. TABLE 1 TNF-α and IFN-γ production by γδT cells BrHpp added TNF-α (pg/ml) IFN-γ (pg/ml) None 12989 ± 1236  98 ±16 200 nM 17406 ± 1290¹ 452 ± 76¹

TABLE 2 Phenotypic Changes of DC upon Coculture with γδ T cells DCcocultures HLA DR CD86 CD83 Alone  645 +/− 324  326 +/− 219  80 +/− 17BrHpp  610 +/− 354  308 +/− 205  69 +/− 14 Activated γδ T cells 1143 +/−549¹  612 +/− 505¹ 140 +/− 35¹ Activated γδ T cells in 1358 +/− 417¹1125 +/− 509¹ 163 +/− 43¹ transwells Activated γδ T cells +  493 +/−269²  261 +/− 150²  66 +/− 19² anti-TNF Ab Activated γδ T cells + 1058+/− 530  533 +/− 439 144 +/− 30 anti-IFNγ Ab

1-64. (Canceled).
 65. A method for the differentiation and/or maturationof immature myeloid dendritic cells (DC) into HLA-DR, CD86, CD83 andIL12 (p40) dendritic cells comprising incubating said DC with δγ Tcells.
 66. The method according to claim 65, wherein said immaturemyeloid DC are derived from monocytes through cytokine treatment chosenfrom IL-4/GM-CSF or IFN-β/IL-3 or functional analogues thereof.
 67. Themethod according to claim 66, wherein said cytokines are addedsimultaneously, sequentially or separately with the δγ T cells to themonocytes.
 68. The method according to claim 65, wherein said δγ T cellsare activated.
 69. The method according to claim 68, wherein saidactivated δγ T cells are produced by treating δγ T cells with anactivating agent chosen from microbial products or derivatives thereof.70. The method according to claim 69, wherein said activating agent isbromohydrin pyrophosphate (BrHPP).
 71. The method according to claim 70wherein BrHPP is present at a concentration of between 10 and 1000 nM.72. The method according to claim 70 wherein BrHPP is present at aconcentration of 200 nM.
 73. A method for obtaining a population ofHLA-DR, CD86, CD83 and IL12 (p40) dendritic cells comprising the stepsof: (a) isolating monocytes from a patient, (b) incubating saidmonocytes in the presence of IFN-β/IL-3, GM-CSF/IL-4 or functionalanalogues thereof, producing a population of immature myeloid dendriticcells, (c) isolating δγ T cells, and, (d) contacting immature myeloiddendritic cells of step (b) with T cells of step (c), whereby saidcontact is performed directly or indirectly.
 74. The method according toclaim 73, wherein the contacting of step (d) is performed for 24 hours.75. The method according to claim 73, wherein said δγ T cells of step(c) are cultured with microbial products or derivatives thereof toactivate said δγ T cells prior to contacting said δγ T cells with saidimmature dendritic cells.
 76. The method according to claim 73, furthercomprising the step of: (e) presenting a peptide on the surface of saiddendritic cells.
 77. The method according to claim 75, furthercomprising the step of: (e) presenting a peptide on the surface of saiddendritic cells.